1. Technical field
The field of this invention is electrophoretic nucleic acid sequencing.
2. Background
Microfluidic devices offer numerous opportunities for rapid manipulations of small volumes. With the ability to move small volumes rapidly from one site to another and perform reactions in very small volumes, where the rate of reaction is greatly enhanced, the opportunities exist to greatly accelerate various operations. However, where the result requires detection of the occurrence of an event or measurement of different species present in a mixture, the detection of a signal from the medium may become the rate-limiting event.
For example, the need to be able to identify a nucleic acid sequence is increasingly important. In research, forensics, identification, as well as for other reasons, one is interested in determining the sequence of a nucleic acid. As the demand for these determinations increases, there is an increasing need for high throughput DNA analyses which provide the sequence with high fidelity. Four-color detection, one color for each nucleotide has become a mainstay of the sequencing process. For reading the colors, various excitation and detection systems have been devised. Each system must accommodate the need to accurately distinguish between background noise and signal and the different wavelengths for the different nucleotides. Furthermore, for rapid determination of the sequence of a large number of nucleic acid samples, one wishes to have numerous channels, with a different sample in each channel being sequenced simultaneously. Such systems exacerbate the problems associated with the various excitation and detection systems.
One of the systems used for nucleic acid sequencing is electrophoresis. The ability to use capillary electrophoresis has greatly improved the opportunities for high throughput sequencing. Initially, arrays of capillaries in close juxtaposition were suggested to be able to do simultaneous determinations. More recently, the opportunity to have a multiplicity of channels in a block of plastic or glass has become available. Depending on the material used for forming the channels, there can be a substantial amount of autofluorescence, which can serve to obscure the signal. In addition to autofluorescence, there is light scatter and cross-talk in the optical system to further limit the sensitivity of detection of the signal. For high throughput, the time of collection of the excitation light is limited and the number of fluorescers in the band being detected is relatively small.
While CCD (charge-coupled devices) detection has many desirable features, such as low cost, ease of use, and large areas of detection, these devices are generally thought to have low sensitivity. The ability to use these detectors where one is detecting fluorescer concentrations of 100 pM or lower at high speed in a multiplex system has been excluded
There is therefore a substantial interest in developing sequencing systems using small devices comprising large numbers of channels so that multiplexed sequence determinations of a large number of samples may be simultaneously, rapidly and accurately performed.
3. Prior Art
U.S. Pat. No. 5,741,411 is an encyclopedic description of the issues associated with capillary electrophoresis and describes the use of CCD devices for detection of multichannel arrays of long capillaries for DNA sequencing. U.S. Pat. No. 5,846,708 describes a multichannel device for detecting molecular tagged targets. Methods for detecting fluorescent signals may be found in U.S. Pat. Nos. 4,820,048; 5,543,026; 5,776,782; and 5,777,733. U.S. Pat. Nos. 5,162,022 and 5,570,015 describe capillary electrophoresis devices in which one or more channels are embodied in a single card.
Methods and devices are provided for rapid, color detection as exemplified by four-color detection in the sequencing of nucleic acids using capillary electrophoresis. The capillary electrophoresis uses a solid substrate comprising a plurality of spaced apart channels with electrodes proximal to the end of each of the channels and a separation medium in each of the channels. Each of the channels is irradiated with a single wavelength excitation light and the emission light divided into four paths by an optical train. The signal in each path is detected with an independent CCD and the signals from the CCD analyzed by a computer. The system provides for accurate resolution of the signals, with greater than 500 nucleotides capable of resolution at high speed.